Expression of Photosynthesis Pathway Gene From Rice and Maize for Understanding Role in Plant Stress and Development Using Bioinformatics Approaches

Rice and maize go to family Poaceae contains many crops of agronomic trait and also represent two carbon metabolism systems, C3 and C4. Analysis of the maize sequence provides new insights into the employment of C3 genes to the C4 mechanism which allowed us to identify more orthologs in other crops. This investigation reports comparative account of genome wide in silico identication of C4 pathway related genes from Zea maize (Zm) and Oryza sativa (Os) from the available whole genome sequence information. The annotation of gene sequences, signature motif analysis, protein phophorylation analysis, study of upstream cis-acting elements, phylogenetic tree construction, chromosomal locations, syntenic mapping and microarray expression analysis of C4 pathway related gene family from both the genomes have been attempted. Results A total of 30 and 37 C4-pathway genes have been predicted from rice and maize genome respectively. Multiple-sequence-alignment and signature motif analysis of these proteins of rice and maize revealed high conserveness. Phophorylation analysis revealed that maize have high number than rice. The phylogenetic analysis of C4 related genes across both plant species clearly resulted in four sub-groups in both plants. In Rice, the 30 genes of C4 pathway related genes family are distributed on eleven out of twelve chromosomes, while in maize, they are randomly distributed on all the chromosomes. Most of the genes of Zm’s chromosome 1 show syntenic relationship with chromosome 1. The cis-regulatory-elements of Zm and Os genes suggested its diverse functions associated with plant growth development, stress and hormone responsiveness as well as endosperm and meristem speci ﬁ c gene expression. This investigation of Zm and Os can now offer new insights into the role of different C4 pathway related genes and examine the comparative syntenic mapping between two monocot models and allows for better understanding about how genes evolve within monocots. Therefore, in silico investigation of C4-photosynthetic-pathway gene family needs to be supported by wet lab experimentation of the novel genes for elucidating their function in many biological courses. of maize (NM_001112268.2) present on Chromosome 6.Cluster B showed relationship between PPDK gene of maize NM_001174252.1 and NM_001148446.1 present on Chromosome 6 and 10 respectively. In PEPCK, Phylogenetic analysis revealed tree to be dividing into two clusters A and B respectively. In cluster A gene of PEPCK of rice (Os03g0255500, Os10g0204400) present on Chromosome 3 and 10 respectively showed synteny with PEPCK gene of maize (NM_001152706.1) present on Chromosome 9 and (NM_001309908.1) for which no matches were found. Cluster B showed a single gene of PEPCK (Os04g0592500) present on Chromosome 4 in relation with Cluster A. In PEPC, Phylogenetic analysis revealed tree to be dividing into two big clusters A and B respectively. Cluster A was further divided into two cluster big a and small b, where cluster a was again divided into one big a1 and one small cluster a2. a1 was further divided into x(x1 and x2) and y clusters. Cluster y revealed PEPC gene of rice (Os02g0244700) present on chromosome 2 in synteny with two isoforms of PEPCK gene of maize (NM_001136893.1, NM_001112033.1) present on Chromosome 4 and 5. Cluster x1 revealed PEPC gene of rice (Os09g0315700) present on Chromosome 9 in synteny with PEPC gene of maize (NM_001111968.1) present on Chromosome 7. In NADP-ME, phylogenetic analysis revealed tree to be dividing into two clusters big A and small B respectively. Cluster A was further divided into one big cluster a1 and one small cluster a2. Cluster a1 into x and y and x into x1 and x2.x1 was further divided into m and n. Cluster x2 revealed relationship between isoform of NADP-ME gene of rice (Os01g0743500) present on Chromosome 1 with isoform of NADP-ME gene of maize (NM_001111822.1) present on Chromosome 8. Cluster x1 revealed 3 relationships involving cluster m1, m2 and n. Cluster m1 revealed relationship between isoform of NADP-ME gene of rice (Os01g0723400) present on Chromosome 1 with isoform of NADP-ME gene of maize (NM_001158924.1) present on Chromosome 3. Cluster m2 revealed relationship between isoform of NADP-ME gene of rice (Os05g0186300) present on Chromosome 5 with isoform of NADP-ME gene of maize (NM_001157493.1) present on Chromosome 6. Cluster n revealed relationship between isoform of NADP-ME gene of rice (Os01g0188400) present on Chromosome 1 with two isoforms of NADP-ME gene of maize (NM_001111843.1, NM_001111913.2) present on Chromosome 3 and 6 respectively. In NADP-MDH, phylogenetic analysis revealed tree to be dividing into two big clusters A and B respectively. Cluster A was further divided into 4 clusters a, b, c and d and cluster B into two x and y respectively. Cluster a1 was further divided into x and y and x into x1 and x2.x1 was further divided into m and n. Cluster a1 revealed relationship between two isoforms of NADP-ME gene of rice (Os01g0649100, Os05g0574400) present on Chromosome 1 and 5 with two isoforms of NADP-ME gene of maize (NM_001148628.2) present on Chromosome 6 and NM_001147865.1 which was not placed. Cluster b1 revealed relationship between isoform of NADP-ME gene of rice (Os12g0632700) present on Chromosome 12 with isoform of NADP-ME gene of maize (NM_001138605.2) present on Chromosome 3. Cluster b2 revealed relationship between isoform of NADP-ME gene of rice (Os03g0773800) present on Chromosome 3 with isoform of NADP-ME gene of maize (NM_001155046.1) present on Chromosome 1. Cluster c revealed relationship between isoform of NADP-ME gene of rice (Os07g0630800) present on Chromosome 7 with isoform of NADP-ME gene of maize (NM_001320799.1) for which no matches were found. Cluster d consisting of d1 and d2 revealed relationship between two isoforms of NADP-ME gene of rice (Os01g0829800, Os08g0434300) present on Chromosome 1 and 8 with three isoforms of NADP-ME gene of maize ( NM_001138830.1, NM_001254820.1 and NM_001138756.1) on Chromosome 1,1 and 4 respectively. Cluster x revealed relationship between isoform of NADP-ME gene of rice (Os08g0562100) present on Chromosome 8 with isoform of NADP-ME gene of maize (NM_001111950.1) present on Chromosome 1. Cluster y1 revealed relationship between isoform of NADP-ME gene of rice (Os08g0562100) present on Chromosome 8 with isoform of NADP-ME gene of maize (NM_001111950.1) present on Chromosome 1. Cluster y2 revealed relationship between isoform of NADP-ME gene of rice (Os10g0478200) present on Chromosome 10 with isoform of NADP-ME gene of maize (NM_001112133.2) present on Chromosome 1 and NM_001153688.1 which was not placed. most expression in NADP-MDH gene (Os03g0773800). GC-motif present in moderate quantity in few genes with most expression in PPDK gene (Os05g0405000) and in Carbonic Anhydrase (Os01g0639900). HSE heat shock element factor involved in heat stress response were present in moderate frequency in few genes with most expression observed in Carbonic Anhydrase (Os11g0153200). LTR and L-box were present in low frequency in only 4 different genes. Box- W1- fungal elicitor responsive element (TTGACC)were present in low frequency in only 3 genes. GCC-Box, WUN-motif and Pc-CMA2a present in only 3, 3 and 2 genes with low frequency. MNF and EIRE were present in low frequency in only single genes. In Maize, Anoxia-response elements (ARE) involved in anaerobic induction was most commonly observed in almost all genes in moderate frequency with higher expression in PPDK gene (ZM2G097457). MYB-binding site MBS) were also present in moderate frequency in almost all genes with higher expression in PEPCK gene (ZM2G001696). TC-rich repeats present in moderate frequency in few genes with most expression in NADP-MDH gene (Zm2G154595). 5 UTR Py-rich Strech were observed in moderate frequency in 4 genes. HSE involved in heat stress response were present in low frequency in only 3 genes. LTR were present in low frequency with exception of NADP-MDH gene (Zm2G068455) with higher expression. L-box was present in low frequency in only single gene. Box- W1- fungal elicitor responsive element (TTGACC) was present in low frequency in only 7 genes. EIRE was present in low frequency in only single genes. MNF was present in low frequency in only 5 genes. ABRE present in higher frequency in only single gene of Carbonic Anhydrase (ZM2G414528). OsPEPC-4, chloroplastic OsCAAlfa-1, glyoxysomal OsNADP-MDH-3 and glyoxysomal OsNADP-MDH-7 were found to be fairly consistent up regulated. OsPPDK-1 was found to be high up regulated at stages which include internode pith parenchyma, root tip, spikelet, embryo and endosperm, which highlights its signicant role in plant nutrition and protection and down regulated at stoma and ovary stage. Chloroplastic OsPPDK-2 was found to give active up regulated expression at callus, suspension cell and dry seed stage. Chloroplastic, NADPME-2 showed overall moderate up regulated expression. Cytoplasmic OsNADPME-3, Chloroplastic OsNADPME-4 and OsNADPME-5 showed overall down regulated expression. Interestingly, OsNADPME-3 exhibit up regulated expression specically at dry seed, embryo-sac and endosperm stage. OsPEPCK-1 showed tissue specic function and was high up regulated for leaf, root, aq leaf and endosperm. OsPEPC-5 was not expressed and show down regulated expression for coleoptiles and germination seed. Chloroplastic OsCABeta-1showed varied expression from down regulated to moderate up regulated expression. OsCA Beta-2,a chloroplastic enzyme, exhibit down regulated expression for developing anther which is crucial reproductive structure. For all other stages the expressions were up regulated. OsCA Alfa-2 exhibit down regulated expression at all anatomical stage. Both glyoxysomal OsNADP MDH-1 and OsNADPMDH-2 showed moderate up regulated expression except for down regulated expression for dry leaf and aq leaf stage respectively. Glyoxysomal OsNADP MDH-4 expressed fairly up regulated but expressed down regulated at whole plant stage. Further study revealed that chloroplastic OsNADP MDH-6 was down regulated at dry seed, coleoptiles and root tip stage where as it showed high up regulated expression for all other specic tissues. Glyoxysomal OsNADP MDH-8 and cytosolic OsNADP MDH-9 showed no signicant Cytosolic OsNADP MDH- 9, overall expression at all stages is down regulated. Os PEPC-4, OsPEPC- 5 showed up regulated expression except for down regulated expression at germinating seedling stage in OsPEPC-5. Chloroplastic OsCA Beta-2 and OsCA Alfa-1 expressions varied from moderate up regulated to high upregulated. P1, P2 and P3 stage was found to be down regulated in OsCA Alfa-1. Further studies revealed that, OsCABeta-1 and Os CA Alfa-2 showed gross down regulated expression. Chloroplastic OsCABeta-1 showed moderate up regulated expression for stages including pre-germination, tillering stage, S1 and S5. It was further observed that, among chloroplast localised enzyme, OsPPDK1 and OsPPDK2, the later showed down regulated expression for 1st leaf, 2nd leaf, 3rd leaf, tiller initiation, tillering stage, P1, P2, P3,P4, P5 stages whereas OsPPDK1 showed fairly high upregulated expression for the same stages. Chloroplastic enzymes, OsNADP ME-1 & OsNADP ME-2, were found to be consistently upregulated except for the down regulated expression at pre-germination stage in OsNADP-ME-2. Further expressions revealed that, cytosolic OsNADP-ME-3 and chloroplast localised OsNADP-ME-4 and OsNADP-ME-5 showed overall expression of down regulated. At Pregermination, S4 and S5 stage upregulated expression was observed in OsNADP- ME-3. OsPEPCK-1 has shown varied expressions. At 2nd leaf, S4 & S5 stage, expressions were observed most up regulated while at callus suspension, pregermination, 3rd leaf, tiller initiation, tillering stage P5, P6, S1, S2 and S3 moderate up regulated expressions were observed.


Background
The prevention of food related consequences arising due to ever increasing population, rapid urbanization, extreme global climate changes and acute water shortage requires an urgent effort to increase the crop yield by a signi cant percentage to meet daily ends of both developing and developed nations. The solution to such a big problem can be developed by multiple approaches. One such approach which seems most promising is introduction of C4 photosynthetic pathways into C3 crops such as rice, which substantially increases the e ciency of plants to utilise nitrogen, water and radiation [1,2], thus producing a higher yield than current C3 crops available [3]. It could be done by detailed analysis of expression pattern of C4 associated genes which in uence biochemical process or produce any prominent morphological and anatomical changes in plants and their protein which could be incorporated into the C3 plants by means of genetic engineering [4,5]. Result from this analysis could be exploited to increase the e ciency of crop yield. Some of the striking similarities that could prove bene cial in future experiments could be relationship between orthologous genes of C4 and C3 plants, changing gene expression patterns of genes in C4 in response to a stimuli which are also present in C3 plants [6]. Incorporating and regulating the expression of C4 genes in C3 plants could also be handled by various approach. C4 pathway is one of three biochemical process used to x atmospheric CO 2 along with CAM & C3 pathway and is characterized by formation of rst 4 carbon product in plants. Also referred as Hatch-Slack Pathway [7].Evolution of C4 pathway has been independently observed 66 times from their C3 ancestors [8], at least in 19 families during angiosperm evolution [9]. C4 pathway has evolved in plants to prevent loss of about 40% of its e ciency which occurs due to photorespiration, an energy demanding process and further more leads to net loss of CO 2 [10].
Photorespiration occurs in response to remove phosphoglycolate-a by product of reaction involving addition of oxygen by Rubisco to ribbulose-1,5 biphosophate resulting in generation of one molecule each of 3-phosphoglycerate and 2-phosphoglycolate [10,11]. Also Phosphoglycolate has no known metabolic purpose and toxic in higher concentrations [11,12]. It has evolved as an adaptation to high temperature and light intensities. C4 plants outperform C3 plants as they use less water per CO 2 xed than C3 plants and hence are more suitable for hot/dry climate [13] Major differences between C4 and C3 pathway involved are:-(1) the differentiation of the two cell and chloroplast types, (2) the presence of an additional set of genes, and (3) a mechanism regulating the cell-speci c expression of these additional genes [14,15]. The enzymes involved in C3 pathway are located in the chloroplast of the bundle sheath cells while those involved in C4 pathway in mesophyll and /or bundle sheath cells for intercellular metabolic cooperation. In NADP-ME pathway it can be seen that PEPCase, NADP-dependent malate dehydrogenase (NADPMDH), and pyruvate phosphate dikinase (PPDK) function in M cells, whereas NADP-ME and RuBPCase activities are localized in BS cells [16,17,18]. The C4 pathway consist of 3 key steps:-A] initial xation of CO 2 into cytosol of mesophyll cells by PEPC [phosphoenolpyruvate carboxylase] to form a C4 acid, Oxaloacetatae [OAA] B] decarboxylation of a C4 acid in bundle sheath cells to release CO 2 and carbon regeneration of primary CO 2 acceptor phosphoenolpyruvate [PEP] [19]. The decarboxylation is catalyzed by 3 enzymes namely NADP-malic enzyme [NADP-ME],NAD malic enzyme [NAD-ME] and phosphoenolpyruvate carboxykinase [PEP-CK]. Recent comparative studies have revealed C3 plants to contain 2 set of genes housekeeping and C4 like genes whose expression level is low in C3 plants. To have a signi cant effect on metabolism gene of C4 plants need to be overexpressed in C3 plants [20]. Studies have shown that the C4 photosynthetic pathway related genes viz. CA, PEPC, NADP-ME, MDH, PPDK and the regulatory protein PPDK-RP in the ag leaf during grain lling stage in different rice genotypes express in C3 rice leaves with a wide genotypic variation [21].
Further studies on the role and contribution of C4 photosynthetic pathway related genes of rice in carbon metabolism will help to enhance the photosynthetic e ciency of rice. Some of the past research indicating effect of overexpressed genes of C4 plants in C3 plants. Our aim was to check role of C4 enzymes individually and to check their role with a combination of other enzymes [22][23]. A series of experiments carried out by researchers in past experiments showed the physiological and photosynthetic effects of C4 enzymes on overexpression in C3 plants. Although PEPC is involved in initial xation of CO 2 in C4 pathway, on overproduction in C3 plants, it stimulated respiration in light and synthesis of amino and organic acids instead of increasing photosynthetic e ciency of rice plants the C4 pathway [24][25]. It has been observed that in the leaves of C3 plants, PEPC has an anaplerotic role replenishing the tricarboxylic acid cycle with intermediates, which are withdrawn for nitrate assimilation and the subsequent amino acid synthesis [26]. No prominent changes were observed in physiological and photosynthetic characteristics of transgenic rice in which C4 speci c PPDK was introduced [27].Although elevation above a threshold level generated a minor response. Overproduction of C4 speci c NADP-ME led to increased photo-inhibition of photosynthesis, leaf chlorophyll bleaching and serious stunting because of increase in the NADPH/NADP ratio in the chloroplast [28].
In the present study, comparative genome wide in silico identi cation of C3/C4 gene to protein such as CA, PEPC, PECK, NADP-ME, NADP-MDH and PPDK involved in photosynthesis pathway of rice and maize. The member further were analyzed in detail in term as chromosomal location(s), gene structure, phylogenetic tree for evolutionary relationship construction and also analyzing the cis-regulatory elements associated with these genes in the promoter region.
Further, comparative phylogeny and syntenic mapping and protein phosphorylation examine with putative protein signature sequences with their function.
Further, we have attempted gene expression analysis development anatomical and stress condition during various stages of panicle and seed development implies their involvement in diverse developmental processes. rice and maize C3/C4 gene family have also been attempted.

Phylogenetic analysis
Amino acid sequences of all the identi ed C3/C4 photosynthetic genes from rice and maize were aligned separately using ClustalW and the phylogenetic tree was constructed using N-J method of MEGA version 4.0.02 [31][32]. Each node was tested using the bootstrap approach by taking 5000 replications to ascertain the reliability of nodes. The number indicated percentages against each node.

Analysis of Signature and Phosphorylation site prediction
To identify the signature sequences within the protein sequences of C3/C4 photosynthetic genes, the deduced protein sequences of all the C3/C4 photosynthetic genes in rice and maize were analyzed using online SMART motifscan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) and ngerPRINTscan (http://www.ebi.ac.uk/Tools/pfa/ ngerprintscan/). Prediction of Serine, threonine and tyrosine speci c Phosphorylation site prediction of C3/C4 protein sequences (Supp Table 1) is done by employing tools NetPhos 2.0 server [33].
Cis-acting regulatory elements/ promoter analysis For promoter analysis, sequences 1,000 bp upstream of the initiation codon of the putative all C3/C4 genes such as CA, PEPC, PEPCK, NADP-ME, NADP-MDH and PPDK were retrieved and subjected to search using CARE program (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/) of Plant CARE database to identify cis-regulatory elements [34]. visualized by Genevestigator. The Expression Omnibus platform accession numbers GSE6901 using for rice. We applied the R package for Quantile normalization of data for removing array to array variation and Z-score conversion for nding out signi cantly expressed gene in a particular tissue. We selected those genes which have z-score > = 1.64 means 90% con dence level. We remove all genes that have standard deviations from the local mean values less than 1.64 for ltering the data. For the cluster analysis of genes related to calcium transporters, we used hierarchical clustering technique using Cluster 3.0. This is a reliable and useful way for analyzing all sorts of large genomic datasets using calculate the distance matrix.

Result And Discussion
Chromosomal Chromosomal distribution and the synteny between rice and maize There is some co-linearity between rice and maize at whole genome level. The orthologs from photosynthetic gene families in both rice and maize were mapped and their corresponding chromosome locations are summarized (Fig. 1A & B). Expansion and inversion of some chromosome region were also revealed during comparative synteny in rice. Most of the genes present on Chromosome 1 of rice showed synteny with genes present on Chromosome 1,3,6,8 and 9. Distribution of photosynthetic genes across chromosome of rice and maize is not uniform but phylogenetic analysis has revealed macro colinearity in between the isoforms of gene to a great degree. Isoforms of genes present on Chromosome 1 of rice showed a higher synteny with genes of maize with beta form of Carbonic Anhydrase [Os01g0639900] of rice present on chromosome 1 depicting synteny with 4 other beta isoforms(NM_001175228.1) present on depicted through chromosomal map. Amino acid sequences of C3 and C4 photosynthetic genes of rice and maize were aligned using Clustal W to study their phylogenetic relationship by constructing rooted (Fig. 2a,b,c,d,e & f) tree using MEGA4. Rooted trees for 6 genes were constructed and analysed for synteny.
Phylogenetic trees of 6 genes showed variance to a great degree. In carbonic anhydrase, Phylogenetic analysis revealed tree to be constructed of two big clusters A (only beta form) and B (only alpha form), where cluster A was subdivided into two small clusters a1 and a2. a1 was further dividing into x and y, while a2 into m and n clusters. Cluster B was dividing into two small cluster b1 and b2. In cluster a1 Beta isoform of CA (Os01g0639900) of rice present on In NADP-ME, phylogenetic analysis revealed tree to be dividing into two clusters big A and small B respectively.
Cluster A was further divided into one big cluster a1 and one small cluster a2. Cluster a1 into x and y and x into x1 and x2.x1 was further divided into m and n.
Cluster x2 revealed relationship between isoform of NADP-ME gene of rice (Os01g0743500) present on Chromosome 1 with isoform of NADP-ME gene of maize (NM_001111822.1) present on Chromosome 8. Cluster x1 revealed 3 relationships involving cluster m1, m2 and n. Cluster m1 revealed relationship between isoform of NADP-ME gene of rice (Os01g0723400) present on Chromosome 1 with isoform of NADP-ME gene of maize (NM_001158924.1) present on Chromosome 3. Cluster m2 revealed relationship between isoform of NADP-ME gene of rice (Os05g0186300) present on Chromosome 5 with isoform of NADP-ME gene of maize (NM_001157493.1) present on Chromosome 6. Cluster n revealed relationship between isoform of NADP-ME gene of rice Evolutionary relationship of C3/C4 photosynthetic pathway genes in rice and maize A rooted tree was constructed to examine evolutionary relationship among C3 and C4 photosynthetic genes of rice and maize respectively by aligning their amino acids though Clustal-W method and tree was constructed using MEGA 4.0 Minimum Evolution method (Fig. 3A). Evolutionary phylogenetic tree analysis revealed tree to be constructed of two big clusters: PEPC gene of rice. Cluster f and g revealed relation between PEPC and NADP-ME of rice and maize respectively Also individual analysis of rice and maize genes was also done by constructing tree respectively by aligning their amino acids through Clustal-W method showed in (Fig. 3B & 3C). Figure 3B depicts evolutionary tree of photosynthetic genes of rice and depict common origin of all photosynthetic genes respectively except that of PEPCK gene (Os04g0592500). Figure 3C depicts evolutionary tree of photosynthetic genes of maize and depict common origin of all photosynthetic genes respectively except that of PPDK gene (NP_001141918.1). Post translational modi cation (phosphorylation) analysis of C3/ C4 protein sequences of rice and maize

Cis-Regulatory Elements Analysis
Graphs of phosphorylation site of 6 photosynthetic genes of C3 and C4 plants were examined and data was analysed and summarized in (Supp Table 2A (Table 3A &  while only one alpha Carbonic Anhydrase showed one common signature while no signature sequences were found in Alpha CA(Os11g0153200). NADP-MDH isoforms showed one common signature sequences to all while only a second tyrosine kinase phosphorylation site was present in (Os01g0829800 and Os08g0562100). While maize, All alpha and beta isoforms of Carbonic Anhydrase had different conserved sequences while no sequences were found for alpha CA (NP_001146392.1). All PEPC isoforms have two signature sequences varying for each gene. All NADP-MDH isoforms have almost single conserved sequence with no signature sequences found for NP_001307728.1. All NADP-ME isoforms have almost two conserved signature sequences with single sequence present in NP_001147966.1. All 9 PEPC isoforms of gene have almost same signature sequences. All two PEPCK isoforms of gene have same signature sequences.
Expression pro ling during plant anatomical stage and across different plant tissues shows differential transcriptional regulation in rice and maize.
In rice, we observed a much diverse pattern of expression of C4 genes during anatomical stage (Fig. 6A). Expression pro ling during plant development and across different plant tissues shows differential transcriptional regulation in rice and maize.
To gain some insights into the possible function of C4 genes in rice, we analyzed the micro-array based expression pattern at different developmental stage (see method). During analysis, we observed a much diverse pattern of expression of C4 genes during all development stages. (Fig. 7A). The enzymes which showed overall down regulated expression throughout the developmental stage studies were OsPEPCK-2, OsPEPCK-3, OsPEPC-1, OsPEPC-2 and OsPEPC3. Interestingly, OsPEPCK-3 showed major involvement only at Panicle 6 and had up regulated expression. OsPEPC-1, OsPEPC-2 and OsPEPC3 genes were expressed in most of the stages with moderate up regulation expression in some speci c stage. Amongst the glyoxysomal enzymes, OsNADP MDH-1, OsNADPMDH-2, OsNADPMDH-3 & OsNADPMDH-4, OsNADPMDH-3 was highly up regulated. OsNADP MDH-1 was moderately down regulated at pregermination stage where as OsNADP MDH-1, OsNADPMDH-2 and OsNADP MDH-4 were found to be moderate up regulated for P1, P2, P3 and P4 stage.
OsNADPMDH-5, a cytosolic enzyme showed most predominantly up regulated expression across all the stages. The expression for OsNADP MDH-6, which is localised in chloroplast, was found to be down regulated at pre-germination stage and moderate down regulated at S5 stage. Other gene enzymes were found to be differentially expressed across the various developmental stages. Among glyoxysomal OsNADP MDH-7 & OsNADP MDH 10, OsNADP MDH − 7 showed up regulated expression at every stage except for S4 and S5 where moderate up regulated expression was found while OsNADP MDH-10 showed moderate down regulated expression for P1, P2, P3 stage and moderated up regulated expression for callus suspension and germination seedling stage which is crucial for initiating development of plant. In Glyoxysomal OsNADP MDH-8 and Cytosolic OsNADP MDH-9, overall expression at all stages is down regulated. Os PEPC-4, OsPEPC-5 showed up regulated expression except for down regulated expression at germinating seedling stage in OsPEPC-5. Chloroplastic OsCA Beta-2 and OsCA Alfa-1 expressions varied from moderate up regulated to high upregulated. P1, P2 and P3 stage was found to be down regulated in OsCA Alfa-1. Further studies revealed that, OsCABeta-1 and Os CA Alfa-2 showed gross down regulated expression. Chloroplastic OsCABeta-1 showed moderate up regulated expression for stages including pre-germination, tillering stage, S1 and S5. It was further observed that, among chloroplast localised enzyme, OsPPDK1 and OsPPDK2, the later showed down regulated expression for 1st leaf, 2nd leaf, 3rd leaf, tiller initiation, tillering stage, P1, P2, P3,P4, P5 stages whereas OsPPDK1 showed fairly high upregulated expression for the same stages. Chloroplastic enzymes, OsNADP ME-1 & OsNADP ME-2, were found to be consistently upregulated except for the down regulated expression at pre-germination stage in OsNADP-ME-2. Further expressions revealed that, cytosolic OsNADP-ME-3 and chloroplast localised OsNADP-ME-4 and OsNADP-ME-5 showed overall expression of down regulated. At Pregermination, S4 and S5 stage upregulated expression was observed in OsNADP-ME-3. OsPEPCK-1 has shown varied expressions. At 2nd leaf, S4 & S5 stage, expressions were observed most up regulated while at callus suspension, pregermination, 3rd leaf, tiller initiation, tillering stage P5, P6, S1, S2 and S3 moderate up regulated expressions were observed.
To gain insights into potential physiological function in Z. mays, we have studied their expression at different stages of plant development and across various plant tissues showed in (Fig. 7B). The enzymes that showed the down regulated expression throughout were chloroplastic enzymes, ZmCA Alpha-2, ZmCA Beta-4, ZmCA Beta-5 and ZmPPDK-2. ZmMDH-4 which is localised in cytosol uniquely showed predominantly up regulated at all developmental stage except for dough stage which is down regulated. ZmCABeta-1 and chloroplastic enzymes ZmCA Beta-2 and ZmCA Beta-3 showed moderate up regulated expression for seedling stage which is important to understand the cotyledon patterns. ZmCABeta-2 exhibit fairly up regulated expression at anthesis and in orescence formation. ZmCA Alpha-1, a chloroplastic enzyme exhibit overall down regulated expression at all developmental stage. Further, chloroplastic ZmMDH-1 was found to exhibit down regulated expression at dough stage, fruit formation and germination stage while ate up regulated expressions were observed for in orescence formation and seedling stage, which holds many important functions during reproduction and provide nutrients to fruits and owers. Further studies revealed that, ZmNADP-ME-1 showed overall down regulated expression except for seedling stage, for which moderate up regulated expression was observed. ZmPEPC-1 and glyoxysomal ZmMDH-2 showed overall moderate down regulated expression at all stages. ZmPEPC-2 expressed up regulated only for seedling stage and its overall expression is down regulated. Further it was observed that ZmNADP ME-2, localised in cytosol, showed varied expressions from down regulated to high regulated. For dough stage and t formation, down regulated expression was observed where as fairly up regulated expression was observed for anthesis and seedling stage. The enzyme expressed most up regulated for in orescence and stem elongationstage. ZmNADPME-3, a chloroplastic enzyme, exhibits high expression for dough stage and fruit formation and was up regulated. ZmPEPCK, glyoxysomal ZmMDH-6 and ZmPEPC-3, showed overall down regulated expression except for seedling stage, dough stage and germination stage, where respectively these enzymes expressed up regulated. The expression exhibit by chloroplastic ZmPPDK-1 was up regulated for dough stage and fruit formation whereas at rest of the stages, its expression was down regulated. The expression of mitochondrial ZmMDH-3, was overall fairly up regulated except for germination stage, where its expression was up regulated the most. Mitochondrial ZmNADP ME-4 was found to be differentially expressed across the various developmental stages. The enzyme did not expressed for dough stage and fruit formation stage and fairly up regulated at stem elongation, seedling stage and germination stage. Notably, it was highly expressed for anthesis and in orescence formation and thus expressed up regulated. ZmMDH-5 which is localised in glyoxysomes consistently expressed down regulated across all developmental stage.

Regulation of Transcription of photosynthetic genes in Rice and Maize under Stress
Analysis of expression of C3 and C4 photosynthetic genes under stress conditions was done using publically available microarray databases result of which is showed in Fig. 8A & 8B.
Expression of maize genes for stress (Water and drought) All 6 isoforms of NADP-MDH gene were downregulated as well as moderate for almost all stress factors. All three isoforms of PEPC gene isoforms were generally downregulated for almost all stress factors with exception for Water (6, 7, 13 and 14) respectively. Single PEPCK gene isoform was downregulated for stress factors (Water1-7) moderate for stress factor (Water 8, 9, 10, 11 and 12) while upregulated for stress factor (Water 13 and 14). Two isoforms of PPDK gene showed moderate and downregulation for various stress factors. All 4 isoforms of NADP-ME gene were downregulated as well as moderate for almost all stress factors. All ve isoforms of beta Carbonic Anhydrase were almost down regulated for all stress factors except Water (13 and 14) while all both isoforms of alpha Carbonic Anhydrasse were also down regulated for all stress factors. Comparative analysis of regulation of photosynthetic genes of rice and maize under stress reveal a total of down regulation of genes of maize in response to stress factors while that of rice was generally upregulated and moderate. This result is corresponding to CARE analysis given in Fig. 4A and B which showed number and frequency of Cis acting regulatory elements high in rice (14 in rice and 11 in maize).CARE such as ARE, MBS, TC-rich repeats, GC motif, HSE were present in higher frequency in rice thus corresponding to the fact of upregulation and moderate expression of C3 photosynthetic genes of rice under stress.

Conclusion
In this investigation, we conducted a comprehensive insilico investigation and identi cation C3/C4 photosynthesis gene/protein sequences in rice and maize.
A complete overview of CA, PEPC, PEPCK, NADPH-ME, NADP-ME and PPDK genes/ protein sequences in rice and maize is presented, including the chromosomal mapping, evolutionary phylogeny relationship, serine, threonine and tyrosine speci c phosphorylation site and protein signature for assessment of posttranslational modi cation and their cis acting regulatory elements analysis. Analysis of protein signature revealed some conserve protein motif present in each protein sequences between rice and maize. Protein phosphorylation analysis clearly revealed role in posttranslational modi cation in C3/C4 genes in both crops but maximum present in maize. The cis-regulatory element analysis of the C3/C4 photosynthesis gene revealed the major putative functions as regulation of genes associated with plant growth development, abiotic and biotic stress, growth hormone and light response. Presence of high number of stress responsive cis acting element in upstream suggests that these proteins might be unregulated in plant stress tolerance. The expression data needs to be correlated in revealing the function of these protein and role in plant stress. A comprehensive analysis on gene expression may provide a better assessment of the prospective genes/ protein for crop improvement. Genome editing based cisgenic approach may be utilized to validate its potential candidature for crop improvement.  Prediction and frequency distribution of Cis-regulatory elements in the upstream region of CA, PEPC, NADP-MDH, NADP-ME, PEPCK, PPDK genes rice and maize Figure 6 Expression pro ling of C3/C4 photosythesis genes of Rice and Maize at different developmental stages using microarray data, Heat map and hierarchical cluster display differential expression pro le for above genes. Various stages are listed in the temporal order of development. The colour bar on top represents relative expression values.

Figure 7
Expression pro ling of C3/C4 photosythesis genes of Rice and Maize at different Anatomical (tissue speci c) using microarray data, Heat map and hierarchical cluster display differential expression pro le for above genes. Different tissue are listed in the temporal order of development. The colour bar on top represents relative expression values.

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